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IntroductionInborn errors of immunity (IEI) are a heterogeneous group of diseases caused by intrinsic defects of the immune system. Estimating the immune competence of immunocompromised patients for an infection risk assessment or after SARS-CoV-2 vaccination constituted a challenge.MethodsThe aim of this study was to determine the humoral responses of patients with IEI through a comprehensive analysis of specific receptor-binding domain-positive (RBD+) IgG+ memory B cells (MBCs) by flow cytometry, together with routine S-specific IgG antibodies and QuantiFERON SARS-CoV-2 (T-cell response), before the vaccine and 3 weeks after a second dose.Results and discussionWe first analyzed the percentage of specific RBD+ IgG+ MBCs in healthy healthcare workers. Within the control group, there was an increase in the percentage of specific IgG+ RBD+ MBCs 21 days after the second dose, which was consistent with S-specific IgG antibodies.Thirty-one patients with IEI were included for the pre- and post-vaccination study; IgG+ RBD+ MBCs were not evaluated in 6 patients due to an absence of B cells in peripheral blood. We detected various patterns among the patients with IEI with circulating B cells (25, 81%): an adequate humoral response was observed in 12/25, consider by the detection of positive S-specific IgG antibodies and the presence of specific IgG+ RBD+ MBCs, presenting a positive T-cell response; in 4/25, very low S-specific IgG antibody counts correlated with undetectable events in the IgG+ RBD+ MBC compartment but with positive cellular response. Despite the presence of S-specific IgG antibodies, we were unable to detect a relevant percentage of IgG+ RBD+ MBCs in 5/25; however, all presented positive T-cell response. Lastly, we observed a profound failure of B and T-cell response in 3 (10%) patients with IEI, with no assessment of S-specific IgG antibodies, IgG+ RBD+ MBCs, and negative cellular response. The identification of specific IgG+ RBD+ MBCs by flow cytometry provides information on different humoral immune response outcomes in patients with IEI and aids the assessment of immune competence status after SARS-CoV-2 mRNA vaccine (BNT162b2), together with S-specific IgG antibodies and T-cell responses.

BackgroundImmune responses to vaccines against severe acute respiratory syndrome (SARS)-coronavirus (CoV)-2 are variable. In the absence of disease, youngsters are expected to better react to vaccines than adults. Nevertheless, chronic immunosuppression in transplant recipients may impair their capability to generate protection. We aim to explore immune responses after BNT162b2 SARS-CoV-2 vaccination in our cohort of young liver-transplanted patients.MethodsA prospective study of adolescent liver-transplanted patients (n=33) in the long-term follow-up was performed. Immune responses after receiving Pfizer-BioNTech BNT162b2 vaccine were analyzed at two time-points: baseline and 30 days after the second dose. Humoral responses were measured by fluoroenzyme-immunoassay and T-cell responses by interferon-γ-release assay. Post-vaccine coronavirus disease (COVID-19) events were recorded by a survey.ResultsPre-vaccine SARS-CoV-2-specific antibodies were undetectable in 27/32 (84.4%), negative/indeterminate in 3/32 (9.4%) and positive in 2/32 (6.3%) patients. Cellular responses at baseline were negative in 12/18 (66.6%), positive in 3/18 (16.6%) and indeterminate in 3/18 (16.6%) recipients. None of the baseline positives recalled any symptoms. Post-vaccine antibodies were detected in all patients and 92.6% showed levels >816 BAU/mL. Twenty (71.4%) recipients had positive T-cell responses. Regarding post-vaccine SARS-Cov-2 infection, 10 (30.3%) patients reported COVID-19 without hospitalization and 21 (63.6%) did not notify any infection. Negative and positive cell-response groups after vaccination showed statistically significant differences regarding COVID-19 cases (62.5% vs 22.2%, respectively; p=0.046).ConclusionsAdolescents and young adults with liver transplantation responded to SARS-Cov-2 vaccine, generating both humoral and cellular responses. Recipients developing cellular responses after vaccination had a lower incidence of COVID-19.

[EN] Primary immunodeficiencies (PIDs) are rare, undiagnosed and potentially fatal diseases. Clinical manifestations of PID can be fatal or leave sequelae that worsen the quality of life of patients. Traditionally, the treatment of PIDs has been largely supportive, with the exception of bone marrow transplantation and, more recently, gene therapy. The discovering of new affected pathways, the development of new molecules and biologics, and the increasing understanding of the molecular basis of these disorders have created opportunities in PIDs therapy. This document aims to review current knowledge and to provide recommendations about the diagnosis and clinical management of adults and children with PIDs based on the available scientific evidence taking in to account current practice and future challenges. A systematic review was conducted, and evidence levels based on the available literature are given for each recommendation where available., [ES] Las inmunodeficiencias primarias (IDP) son unas enfermedades raras, frecuentemente infradiagnosticadas y potencialmente fatales. Las manifestaciones clínicas de las IDP pueden ser muy graves y ocasionar secuelas que empeoran la calidad de vida de los pacientes. Tradicionalmente, el tratamiento de las IDP ha sido fundamentalmente de soporte, con excepción del trasplante de progenitores hematopoyéticos y, más recientemente, la terapia génica. El descubrimiento de nuevos mecanismos patogénicos, el desarrollo de nuevas moléculas y fármacos biológicos y los avances en el conocimiento de las bases moleculares de estas enfermedades han abierto oportunidades para el tratamiento de esta afección. El objetivo de este documento es revisar el conocimiento actual y aportar recomendaciones para el diagnóstico y el tratamiento clínico de los pacientes adultos y pediátricos con IDP basado en la evidencia científica disponible y teniendo en cuenta la actual práctica y los retos futuros. Se realizó una revisión sistemática, que justifica los niveles de evidencia para cada recomendación.

Copyright © 2021 del Pino-Molina, López-Granados, Lecrevisse, Torres Canizales, Pérez-Andrés, Blanco, Wentink, Bonroy, Nechvatalova, Milota, Kienzler, Philippé, Sousa, van der Burg, Kalina, van Dongen and Orfao. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms., Introduction: Common Variable Immunodeficiency (CVID) is characterized by defective antibody production and hypogammaglobulinemia. Flow cytometry immunophenotyping of blood lymphocytes has become of great relevance for the diagnosis and classification of CVID, due to an impaired differentiation of mature post-germinal-center (GC) class-switched memory B-cells (MBC) and severely decreased plasmablast/plasma cell (Pb) counts. Here, we investigated in detail the pre-GC B-cell maturation compartment in blood of CVID patients. Methods: In this collaborative multicentric study the EuroFlow PID 8-color Pre-GC B-cell tube, standardized sample preparation procedures (SOPs) and innovative data analysis tools, were used to characterize the maturation profile of pre-GC B-cells in 100 CVID patients, vs 62 age-matched healthy donors (HD). Results: The Pre-GC B-cell tube allowed identification within pre-GC B-cells of three subsets of maturation associated immature B-cells and three subpopulations of mature naïve B-lymphocytes. CVID patients showed overall reduced median absolute counts (vs HD) of the two more advanced stages of maturation of both CD5+ CD38+/++ CD21het CD24++ (2.7 vs 5.6 cells/µl, p=0.0004) and CD5+ CD38het CD21+ CD24+ (6.5 vs 17 cells/µl, p1 (CD38, CD5, CD19, CD21, CD24, and/or smIgM) phenotypic marker (57/88 patients; 65%) for a total of 3 distinct CVID patient profiles (group 1: 42/88 patients, 48%; group 2: 8/88, 9%; and group 3: 7/88, 8%) and ii) CVID patients with a clearly altered pre-GC B cell maturation pathway in blood (group 4: 31/88 cases, 35%). Conclusion: Our results show that maturation of pre-GC B-cells in blood of CVID is systematically altered with up to four distinctly altered maturation profiles. Further studies, are necessary to better understand the impact of such alterations on the post-GC defects and the clinical heterogeneity of CVID., The coordination and innovation processes of this study were supported by the EuroFlow Consortium (Chairmen: MB and AO). LP-M was supported by FIS PI16/01605 and JTC by FIS PI13/02296 (Fondo de Investigación Sanitaria Instituto de Salud Carlos III, Madrid, Spain). The work was partially supported by grant PI20/01712-FEDER (Fondo de Investigación Sanitaria Instituto de Salud Carlos III, Madrid, Spain) and a grant from Fundación Mutua Madrileña (MMA, Madrid, Spain).

We read with interest the recent articles from Zhang et al and Bastard et al revealing impaired type I IFN immunity as a major risk for severe coronavirus disease 2019 (COVID-19)1,2; however, the relative importance of innate and adaptive immunity for viral control is difficult to evaluate.3 A diagnosis of mild COVID-19 in 2 identical twin neonates with an uncommon primary immunodeficiency (PID) that causes athymia and almost complete T-cell lymphopenia allowed study of innate immunity to severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection in the absence of adaptive immunity.

BACKGROUND: There is uncertainty about the impact of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection in individuals with rare inborn errors of immunity (IEI), a population at risk of developing severe coronavirus disease 2019. This is relevant not only for these patients but also for the general population, because studies of IEIs can unveil key requirements for host defense. OBJECTIVE: We sought to describe the presentation, manifestations, and outcome of SARS-CoV-2 infection in IEI to inform physicians and enhance understanding of host defense against SARS-CoV-2. METHODS: An invitation to participate in a retrospective study was distributed globally to scientific, medical, and patient societies involved in the care and advocacy for patients with IEI. RESULTS: We gathered information on 94 patients with IEI with SARS-CoV-2 infection. Their median age was 25 to 34 years. Fifty-three patients (56%) suffered from primary antibody deficiency, 9 (9.6%) had immune dysregulation syndrome, 6 (6.4%) a phagocyte defect, 7 (7.4%) an autoinflammatory disorder, 14 (15%) a combined immunodeficiency, 3 (3%) an innate immune defect, and 2 (2%) bone marrow failure. Ten were asymptomatic, 25 were treated as outpatients, 28 required admission without intensive care or ventilation, 13 required noninvasive ventilation or oxygen administration, 18 were admitted to intensive care units, 12 required invasive ventilation, and 3 required extracorporeal membrane oxygenation. Nine patients (7 adults and 2 children) died. CONCLUSIONS: This study demonstrates that (1) more than 30% of patients with IEI had mild coronavirus disease 2019 (COVID-19) and (2) risk factors predisposing to severe disease/mortality in the general population also seemed to affect patients with IEI, including more younger patients. Further studies will identify pathways that are associated with increased risk of severe disease and are nonredundant or redundant for protection against SARS-CoV-2. ispartof: JOURNAL OF ALLERGY AND CLINICAL IMMUNOLOGY vol:147 issue:2 pages:520-531 ispartof: location:United States status: published

Use of adoptive immunotherapy with virus-specific T cells (VST) in patients with inborn errors of immunity prior to hematopoietic stem cell transplantation (HSCT) has been reported in few patients. We report our experience, reviewing all the cases previously reported. We report four children with inborn errors of immunity who received VST infusion in a pre-HSCT setting in two reference centers in Spain and review all inborn errors of immunity cases previously reported. Taking into account our four cases, nine children have been reported to receive VST prior to HSCT to date: 3 severe combined immunodeficiency, 2 CTPS1 deficiency, 1 dyskeratosis congenital, 1 ORAI1 deficiency, 1 Rothmund-Thomson syndrome, and 1 combined immunodeficiency without confirmed genetic defect. In four patients, immunotherapy resulted in clinical improvement, allowing to proceed to HSCT. In these cases, the infusion was started closely to viral diagnosis [mean time 28 days (IQR; 17–52 days)], and the VST was followed shortly thereafter by HSCT [mean time 28 days (IQR; 10–99 days)]. Viremia was controlled after HSCT in two cases (performed 7 and 36 days after the infusion). Multiple infusions were required in many cases. Five out of nine patients died before receiving HSCT. These patients presented with a prolonged and uncontrolled infection before VST administration [mean time from viral diagnosis to VST infusion was 176 days (IQR; 54–1687)]. In patients with inborn errors of immunity, the efficacy of VST for treating disseminated viral infections in pre-transplant settings seems to have a limited efficacy. However, this therapy could be used in a pre-emptive setting before severe viral disease occurs or closely to HSCT.

Mutations in PIK3R1 gene have been associated to two different conditions: a primary immunodeficiency, called APDS2, of recent description and SHORT syndrome. 47 patients with APDS2 have been reported to date, only one of them sharing both PIK3R1-related phenotypes. Here we describe two more patients affected by APDS2 and SHORT syndrome, which highlights that this association may not be so infrequent. We recommend that patients with mutations in PIK3R1 gene should be assessed by both clinical immunologists and clinical geneticists.

During the clinical workup of an adolescent male with chronic active EBV disease, it was noted that natural killer (NK) cells of this individual did not express the Fc-receptor CD16A (Fig 1, A). The index patient, born to nonconsanguineous parents, was first seen when he was 11 years old, and an extended workup revealed persistently high levels of EBV-encoded small RNA (EBER) DNA in peripheral blood and multiple-organ infiltration with EBV-positive T lymphocytes including an IgA- and EBV-positive interstitial nephritis that eventually led to renal failure (see Case Report and Table E1 in this article’s Online Repository at www.jacionline.org)., This work was supported by grants SAF2014-58752-R and SAF2017-83265-R to H.T.R. and grant SAF2016-80363-C2-2-R to C.V. (Spanish Science agency/European Regional Development Fund, European Union, AEI/FEDER, EU). The group of M.V.-G. has also received funding from the Spanish Ministry of Science and Innovation (grant nos. RTC-2017-6379-1 and RTI2018-093569-B-I00 [MCIU/AEI/FEDER, EU]) and the regional government of Madrid (grant no. S2017/BMD-3733-2). The group of M.V.-G. also belongs to the research network TENTACLES (RED2018-102411-T) funded by the Spanish Ministry of Science. A.P.P. and A.B.M. acknowledge PhD studentships funded by the National Secretary of Higher Education, Science, Technology and Innovation (SENESCYT, Ecuador) and Spanish Ministry of Economy and Competitivity (MINECO), respectively (SVP-2014-068263). M.M. was supported by grant GCB15152947MELE from Fundación de la Asociación Española contra el Cáncer. Work in the laboratory of H.H. is supported by Infect-ERA grant TANKACY, BMBF 031L0090, and German Research Foundation (DFG) grant HE2526/9-1. E.L.G. has grants from the Spanish Association Against Cancer (AECC) and Carlos III Health Institute (grant no. PI16/01605). C.A.B. acknowledges support from the National Institutes of Health (grant no. DP2AI11219301).

Currently, there is a need for reliable tests that allow identification of individuals that have been infected with SARS-CoV-2 even if the infection was asymptomatic. To date, the vast majority of the serological tests for SARS-CoV-2–specific Abs are based on serum detection of Abs to either the viral spike glycoprotein (the major target for neutralizing Abs) or the viral nucleocapsid protein that is known to be highly immunogenic in other coronaviruses. Conceivably, exposure of Ags released from infected cells could stimulate Ab responses that might correlate with tissue damage and, hence, they may have some value as a prognostic indicator. We addressed whether other nonstructural viral proteins, not incorporated into the infectious viral particle, specifically the viral cysteine-like protease, might also be potent immunogens. Using ELISA tests, coating several SARS-CoV-2 proteins produced in vitro, we describe that COVID-19 patients make high titer IgG, IgM, and IgA Ab responses to the Cys-like protease from SARS-CoV-2, also known as 3CLpro or Mpro, and it can be used to identify individuals with positive serology against the coronavirus. Higher Ab titers in these assays associated with more-severe disease, and no cross-reactive Abs against prior betacoronavirus were found. Remarkably, IgG Abs specific for Mpro and other SARS-CoV-2 Ags can also be detected in saliva. In conclusion, Mpro is a potent Ag in infected patients that can be used in serological tests, and its detection in saliva could be the basis for a rapid, noninvasive test for COVID-19 seropositivity., This work was supported by the Spanish National Research Council (Project 202020E079) and grants from the Madrid Regional Government (IMMUNOTHERCAN S2017/BMD-3733-2 [to M.V.-G.]), the Spanish Ministry of Science and Innovation (MCIU/AEI/FEDER, EU: RTI2018-093569-B-I00 [to M.V.-G.], SAF2017-82940-R [to J.M.R.F.], SAF2017-83265-R [to H.T.R.], and SAF2017-82886-R [to F.S.-M.]), and RETICS Program of Instituto de Salud Carlos III (RD16/0012/0006; RIER [to J.M.R.F.], RD16/0011/0012 and PI18/0371 [to I.G.-A.], and PI19/00549 [to A.A.]). The study was also funded by La Caixa Banking Foundation (HR17-00016 to F.S.-M.) and Banco Santander Supera COVID to F.S.-M. This work has been cofunded by grant Covid 2019 from the Madrid Regional Government to Health Institute “La Princesa.”

Autoimmune lymphoproliferative syndrome is a primary immunodeficiency caused by variants in FAS-mediated apoptosis related genes and is characterized by lymphadenopathy, splenomegaly and autoimmunity. A total of six different variants in CASP10 have been described as potential causative of disease, although two of them have recently been considered polymorphisms. The high allele frequency of these variants in healthy population in addition to the broad clinical spectrum of the disease difficult the interpretation of their pathogenicity. Here, we describe the clinical and analytical findings of three new patients carrying variants in CASP10 and summarize 12 more cases from the literature. Autoimmune cytopenias, adenopathies and increment of TCRαβ+CD4-CD8- cells have been the most common findings, being possibly the FAS-mediated apoptosis pathway the pathogenic mechanism of this disease. The clinical impact and the consequences of CASP10 variants are not fully elucidated, therefore the description of new cases will contribute to solve this issue.

Primary immunodeficiencies (PIDs) are immune disorders resulting from defects in genes involved in immune regulation, and manifesting as an increased susceptibility to infections, autoimmunity, and cancer. However, the molecular basis of some prevalent entities remains poorly understood. Epigenetic control is essential for immune functions, and epigenetic alterations have been identified in different PIDs, including syndromes such as immunodeficiency-centromeric-instability-facial-anomalies, Kabuki, or Wolf–Hirschhorn, among others. Although the epigenetic changes may differ among these PIDs, the reversibility of epigenetic modifications suggests that they might become potential therapeutic targets. Here, we review recent mechanistic advances in our understanding of epigenetic alterations associated with certain PIDs, propose that a fully epigenetically driven mechanism might underlie some PIDs, and discuss the possible prophylactic and therapeutic implications., FEDER, the ‘Fondo de Investigaciones Sanitarias/Instituto de Salud Carlos III’ (PI14/ 00025), MINECO (SAF2017-83061-R), the ‘Fundación Ramón Areces’, the ‘Federación Española de Enfermedades Raras’, the ‘Asociación Muévete por los que no Pueden’, and a Research Contract with the ‘Fundación Síndrome de Wolf- Hirschhorn o 4p-’. Institutional grants from the ‘Fundación Ramón Areces’ and ‘Banco de Santander’ to the CBMSO

The clinical value of assessing immunoglobulin (Ig)G and IgA subclasses in addition to the isotypes of soluble Igs in serum has been well established. > 20 years ago, the International Union of Immunological Societies and the World Health Organization performed collaborative studies in order to validate antibody (Ab) clones for the detection of IgG and IgA subclasses for a broad range of laboratory assays, except for flow cytometry. Here we analyzed the performance of commercially available Ab clones to detect IgG and IgA subclasses in memory B-cells and plasma cells (PCs) by flow cytometry. In a first step, 28 Ab clones were evaluated in peripheral blood from healthy donors. Only 17/28 clones showed reactivity against IgG and IgA subclasses expressed on the B-cell and PC surface membrane, including Ab clones for IgG1 (SAG1, HP6188, HP6001 and HP6186), IgG2 (SAG2, HP6014 and HP6002), IgG3 (SAG3, HP6095 and HP6050), IgG4 (SAG4), IgA1 (SAA1, H69-11.4 and B3506B4) and IgA2 (SAA2, 2E2, and A9604D2). In a second step, for each Ig subclass a single clone was selected according to its specificity and fluorescence intensity (resolution power), for further more detailed validation (SAG1, SAG2, SAG3, SAG4, SAA1 and SAA2). This validation process was carried out in 4 different laboratories by testing the selected Ab clones in human peripheral blood, bone marrow and tonsil samples, using different staining protocols (e.g. surface membrane and/or cytoplasmic staining). All selected Ab clones displayed strong positivity, high specificity and optimal resolution between negative and positive cells. Alternative Ab clones were also validated. Thus, our results show the feasibility of using the validated Ig subclass Ab clones in combination with other B cell-associated markers for detailed dissection of the memory B-cell and PC compartments that express distinct Ig subclasses in different human tissues., EB is supported by a grant from the Junta de Castilla y León (Fondo Social Europeo, ORDEN EDU/346/2013, Valladolid, Spain). This work was supported by the: CB16/12/00400 grant (CIBER/ONC, Instituto de Salud Carlos III, Ministerio de Economía y Competitividad, -Madrid, Spain- and FONDOS FEDER) and the FIS PI12/00905-FEDER grant, from the Fondo de Investigación Sanitaria of Instituto de Salud Carlos III, Madrid, Spain)

14 p.-5 fig.-5 tab., Supported by grants SAF2014-58752-R and SAF2017-83265-R (to H.T.R.), SAF2017-83785-R (to A.D.-S.; MINECO/AEI/FEDER, UE), and a PhD studentship to A.B.M. (MINECO, SVP-2014-068263). E.L.-G. has grants from the Spanish Association Against Cancer (AECC) and Carlos III Health Institute PI16/01605).

Funding: E.B. was supported by a grant from the Junta de Castilla y Leon (Fondo Social Europeo, ORDEN EDU/346/2013, Valladolid, Spain). This work was supported by the CB16/ 12/00400 and CB/16/12/00233 grants (CIBERONC, Instituto de Salud Carlos III, Ministerio de Economıa y Competitividad, Madrid, Spain, and FONDOS FEDER), the FIS PI12/00905-FEDER grant (Fondo de Investigacion Sanitaria of Instituto de Salud Carlos III, Madrid, Spain) and a grant from Fundacion Mutua Madrile~na (Madrid, Spain). The coordination and innovation processes of this study were supported by the EuroFlow Consortium. Background: Predominantly antibody deficiencies (PADs) are the most prevalent primary immunodeficiencies, but their B-cell defects and underlying genetic alterations remain largely unknown. Objective: We investigated patients with PADs for the distribution of 41 blood B-cell and plasma cell (PC) subsets, including subsets defined by expression of distinct immunoglobulin heavy chain subclasses. Methods: Blood samples from 139 patients with PADs, 61 patients with common variable immunodeficiency (CVID), 68 patients with selective IgA deficiency (IgAdef), 10 patients with IgG subclass deficiency with IgA deficiency, and 223 age-matched control subjects were studied by using flow cytometry with EuroFlow immunoglobulin isotype staining. Patients were classified according to their B-cell and PC immune profile, and the obtained patient clusters were correlated with clinical manifestations of PADs. Results: Decreased counts of blood PCs, memory B cells (MBCs), or both expressing distinct IgA and IgG subclasses were identified in all patients with PADs. In patients with IgAdef, B-cell defects were mainly restricted to surface membrane (sm)IgA + PCs and MBCs, with 2 clear subgroups showing strongly decreased numbers of smIgA + PCs with mild versus severe smIgA + MBC defects and higher frequencies of nonrespiratory tract infections, autoimmunity, and affected family members. Patients with IgG subclass deficiency with IgA deficiency and those with CVID showed defects in both smIgA + and smIgG + MBCs and PCs. Reduced numbers of switched PCs were systematically found in patients with CVID (absent in 98%), with 6 different defective MBC (and clinical) profiles: (1) profound decrease in MBC numbers; (2) defective CD27 + MBCs with almost normal IgG 3 + MBCs; (3) absence of switched MBCs; and (4) presence of both unswitched and switched MBCs without and; (5) with IgG 2 + MBCs; and (6) with IgA 1 + MBCs. Conclusion: Distinct PAD defective B-cell patterns were identified that are associated with unique clinical profiles. publishersversion published

Copyright © 2019 van der Burg, Kalina, Perez-Andres, Vlkova, Lopez-Granados, Blanco, Bonroy, Sousa, Kienzler, Wentink, Mejstríková, Šinkorova, Stuchly, van Zelm, Orfao and van Dongen. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms., In the rapidly evolving field of primary immunodeficiencies (PID), the EuroFlow consortium decided to develop a PID orientation and screening tube that facilitates fast, standardized, and validated immunophenotypic diagnosis of lymphoid PID, and allows full exchange of data between centers. Our aim was to develop a tool that would be universal for all lymphoid PIDs and offer high sensitivity to identify a lymphoid PID (without a need for specificity to diagnose particular PID) and to guide and prioritize further diagnostic modalities and clinical management. The tube composition has been defined in a stepwise manner through several cycles of design-testing-evaluation-redesign in a multicenter setting. Equally important appeared to be the standardized pre-analytical procedures (sample preparation and instrument setup), analytical procedures (immunostaining and data acquisition), the software analysis (a multidimensional view based on a reference database in Infinicyt software), and data interpretation. This standardized EuroFlow concept has been tested on 250 healthy controls and 99 PID patients with defined genetic defects. In addition, an application of new EuroFlow software tools with multidimensional pattern recognition was designed with inclusion of maturation pathways in multidimensional patterns (APS plots). The major advantage of the EuroFlow approach is that data can be fully exchanged between different laboratories in any country of the world, which is especially of interest for the PID field, with generally low numbers of cases per center., The coordination and innovation processes of this study were supported by the EuroFlow Consortium (Chairmen: MvdB and AO). MvZ is supported by Senior Research Fellowship GNT1117687 from the Australian National Health and Medical Research Council. TK and EM were supported by projects 15-28541A from Ministry of Health, LO1604 from Ministry of Education, Youth and Sports and GBP302/12/G101 from Grant Agency of the Czech Republic. MP-A, EB, and AO were supported by a grant from the Junta de Castilla y León (Fondo Social Europeo, ORDEN EDU/346/2013, Valladolid, Spain) and the CB16/12/00400 grant (CIBER/ONC, Instituto de Salud Carlos III, Ministerio de Economía y Competitividad, - Madrid, Spain- and FONDOS FEDER), the FIS PI12/00905-FEDER grant (Fondo de Investigación Sanitaria of Instituto de Salud Carlos III, Madrid, Spain) and AP119882013 grant (Fundación Mutua Madrileña, Madrid, Spain). Publishing costs for this article were covered by the International Union of Immunological Societies (IUIS).

BACKGROUND: Postzygotic de novo mutations lead to the phenomenon of gene mosaicism. The 3 main types are called somatic, gonadal, and gonosomal mosaicism, which differ in terms of the body distribution of postzygotic mutations. Mosaicism has been reported occasionally in patients with primary immunodeficiency diseases (PIDs) since the early 1990s, but its real involvement has not been systematically addressed. OBJECTIVE: We sought to investigate the incidence of gene mosaicism in patients with PIDs. METHODS: The amplicon-based deep sequencing method was used in the 3 parts of the study that establish (1) the allele frequency of germline variants (n = 100), (2) the incidence of parental gonosomal mosaicism in families with PIDs with de novo mutations (n = 92), and (3) the incidence of mosaicism in families with PIDs with moderate-to-high suspicion of gene mosaicism (n = 36). Additional investigations evaluated body distribution of postzygotic mutations, their stability over time, and their characteristics. RESULTS: The range of allele frequency (44.1% to 55.6%) was established for germline variants. Those with minor allele frequencies of less than 44.1% were assumed to be postzygotic. Mosaicism was detected in 30 (23.4%) of 128 families with PIDs, with a variable minor allele frequency (0.8% to 40.5%). Parental gonosomal mosaicism was detected in 6 (6.5%) of 92 families with de novo mutations, and a high incidence of mosaicism (63.9%) was detected among families with moderate-to-high suspicion of gene mosaicism. In most analyzed cases mosaicism was found to be both uniformly distributed and stable over time. CONCLUSION: This study represents the largest performed to date to investigate mosaicism in patients with PIDs, revealing that it affects approximately 25% of enrolled families. Our results might have serious consequences regarding treatment and genetic counseling and reinforce the use of next-generation sequencing-based methods in the routine analyses of PIDs. ispartof: JOURNAL OF ALLERGY AND CLINICAL IMMUNOLOGY vol:143 issue:1 pages:359-368 ispartof: location:United States status: published

Guidelines for screening for primary immunodeficiencies (PID) are well-defined and several consensus diagnostic strategies have been proposed. These consensus proposals have only partially been implemented due to lack of standardization in laboratory procedures, particularly in flow cytometry. The main objectives of the EuroFlow Consortium were to innovate and thoroughly standardize the flowcytometric techniques and strategies for reliable and reproducible diagnosis and classification of PID of the lymphoid system. The proposed EuroFlow antibody panels comprise one orientation tube and seven classification tubes and corresponding databases of normal and PID samples. The 8-color 12-antibody PID Orientation tube (PIDOT) aims at identification and enumeration of the main lymphocyte and leukocyte subsets; this includes naive pre-germinal center (GC) and antigen-experienced post-GC memory B-cells and plasmablasts. The seven additional 8(-12)-color tubes can be used according to the EuroFlow PID algorithm in parallel or subsequently to the PIDOT for more detailed analysis of B-cell and T-cell subsets to further classify PID of the lymphoid system. The Pre-GC, Post-GC, and immunoglobulin heavy chain (IgH)-isotype B-cell tubes aim at identification and enumeration of B-cell subsets for evaluation of B-cell maturation blocks and specific defects in IgH-subclass production. The severe combined immunodeficiency (SCID) tube and T-cell memory/effector subset tube aim at identification and enumeration of T-cell subsets for assessment of T-cell defects, such as SCID. In case of suspicion of antibody deficiency, PIDOT is preferably directly combined with the IgH isotype tube(s) and in case of SCID suspicion (e.g., in newborn screening programs) the PIDOT is preferably directly combined with the SCID T-cell tube. The proposed >= 8-color antibody panels and corresponding reference databases combined with the EuroFlow PID algorithm are designed to provide fast, sensitive and cost-effective flowcytometric diagnosis of PID of the lymphoid system, easily applicable in multicenter diagnostic settings world-wide.

Common Variable Immunodeficiency (CVID) is characterized by impaired antibody production and poor terminal differentiation of the B cell compartment, yet its pathogenesis is still poorly understood. We first reported the occurrence of epigenetic alterations in CVID by high-throughput methylation analysis in CVID-discordant monozygotic twins. Data from a recent whole DNA methylome analysis throughout different stages of normal B cell differentiation allowed us to design a new experimental approach. We selected CpG sites for analysis based on two criteria: one, CpGs with potential association with the transcriptional status of relevant genes for B cell activation and differentiation; and two, CpGs that undergo significant demethylation from naive to memory B cells in healthy individuals. DNA methylation was analyzed by bisulfite pyrosequencing of specific CpG sites in sorted naive and memory B cell subsets from CVID patients and healthy donors. We observed impaired demethylation in two thirds of the selected CpGs in CVID memory B cells, in genes that govern B cell-specific processes or participate in B cell signaling. The degree of demethylation impairment associated with the extent of the memory B cell reduction. The impaired demethylation in such functionally relevant genes as AICDA in switched memory B cells correlated with a lower proliferative rate. Our new results reinforce the hypothesis of altered demethylation during B cell differentiation as a contributing pathogenic mechanism to the impairment of B cell function and maturation in CVID. In particular, deregulated epigenetic control of AICDA could play a role in the defective establishment of a post-germinal center B cell compartment in CVID.

Primary antibody deficiencies (PADs), the most prevalent inherited primary immunodeficiencies (PIDs), are associated with a wide range of genetic alterations (both monogenic or polygenic) in B cell-specific genes. However, correlations between the genotype and clinical manifestations are not evident in all cases indicating that genetic interactions, environmental and epigenetic factors may have a role in PAD pathogenesis. The recent identification of key defects in DNA methylation in common variable immunodeficiency as well as the multiple evidences on the role of epigenetic control during B cell differentiation, activation and during antibody formation highlight the importance of investing research efforts in dissecting the participation of epigenetic defects in this group of diseases. This review focuses on the role of epigenetic control in B cell biology which can provide clues for the study of potential novel pathogenic defects involved in PADs.

Common variable immunodeficiency (CVID) is the most frequent symptomatic primary immunodeficiency characterized by recurrent infections, hypogammaglobulinemia and poor response to vaccines. Its diagnosis is made based on clinical and immunological criteria, after exclusion of other diseases that can cause similar phenotypes. Currently, less than 20% of cases of CVID have a known underlying genetic cause. We have analyzed whole-exome sequencing and copy number variants data of 36 children and adolescents diagnosed with CVID and healthy relatives to estimate the proportion of monogenic cases. We have replicated an association of CVID to p.C104R in TNFRSF13B and reported the second case of homozygous patient to date. Our results also identify five causative genetic variants in LRBA, CTLA4, NFKB1, and PIK3R1, as well as other very likely causative variants in PRKCD, MAPK8, or DOCK8 among others. We experimentally validate the effect of the LRBA stop-gain mutation which abolishes protein production and downregulates the expression of CTLA4, and of the frameshift indel in CTLA4 producing expression downregulation of the protein. Our results indicate a monogenic origin of at least 15-24% of the CVID cases included in the study. The proportion of monogenic patients seems to be lower in CVID than in other PID that have also been analyzed by whole exome or targeted gene panels sequencing. Regardless of the exact proportion of CVID monogenic cases, other genetic models have to be considered for CVID. We propose that because of its prevalence and other features as intermediate penetrancies and phenotypic variation within families, CVID could fit with other more complex genetic scenarios. In particular, in this work, we explore the possibility of CVID being originated by an oligogenic model with the presence of heterozygous mutations in interacting proteins or by the accumulation of detrimental variants in particular immunological pathways, as well as perform association tests to detect association with rare genetic functional variation in the CVID cohort compared to healthy controls. This study was funded by grants SAF2012-35025 and SAF2015-68472-C2-2-R from the Ministerio de Economía y Competitividad (Spain) and FEDER (EU) to FC; by Direcció General de Recerca, Generalitat de Catalunya (2014SGR-866 and 2017SGR-702) to FC and EB; to EB by grant BFU2016-77961-P from Ministerio de Economía, Industria y Competitividad (Spain) AEI (Spain) and FEDER (EU); by Instituto de Salud Carlos III, grant PI14/00405, cofinanced by the European Regional Development Fund (ERDF) to RC; partially funded by CERCA Programme/Generalitat de Catalunya (JIA), and SAF2015-68472-C2-1-R grant from the Spanish Ministry of Economy and Competitiveness co-financed by European Regional Development Fund (ERDF) to JIA; GV-I was supported by grant BES-2012-051794; JH-R was supported by grant BES-2013-064333. TMB is supported by U01 MH106874 grant, Howard Hughes International Early Career, Obra Social “La Caixa” and Secretaria d'Universitats i Recerca del Departament d'Economia i Coneixement de la Generalitat de Catalunya. All phases of this study were supported by the projects PI12/01990 and PI15/01094 to LA and PI13/00676 to MJ. This work was also supported by the Jeffrey Modell Foundation. This study makes use of data generated by the Medical Genome Project. A full list of the investigators who contributed to the generation of the data is available from http://www.medicalgenomeproject.com/en. Funding for the project was provided by the Spanish Ministry of Economy and Competitiveness, projects I + D + i 2008, Subprograma de actuaciones Científicas y Tecnológicas en Parques Científicos y Tecnológicos (ACTEPARQ 2009) and ERFD.

Background CD3δ deficiency is a fatal form of severe combined immunodeficiency that can be cured by hematopoietic stem cell transplantation (HSCT). The presence of a thymus loaded with T-cell progenitors in patients with CD3δ deficiency may require special considerations in choosing the regimen of conditioning and the type of HSCT. Objectives To study the outcome of CD3δ deficiency by using various modalities of stem cell transplantation. Methods We analyzed data on 13 patients with CD3δ deficiency who underwent HSCT in 7 centers. HSCT was performed by using different sources of donor stem cells as well as various conditioning regimens. Results One patient received stem cells from a matched related donor and survived after a second transplant, needing substantial conditioning in order to engraft. Only 2 of 7 other patients who received a mismatched related donor transplant survived; 2 of them had no conditioning, whereas the others received various combinations of conditioning regimens. Engraftment of T cells in the survivors appears incomplete. Three other patients who received stem cells from a matched unrelated donor survived and enjoyed full immune reconstitution. Two patients received unrelated cord blood without conditioning. One of them has had a partial but stable engraftment, whereas the other engrafted well but is only 12 months after HSCT. We also report here for the first time that patients with CD3δ deficiency can present with typical features of Omenn syndrome. Conclusions HSCT is a successful treatment for patients with CD3δ deficiency. The small number of patients in this report prevents definitive statements on the importance of survival factors, but several are suggested: (1) HLA-matched donor transplants are associated with superior reconstitution and survival than are mismatched donor transplants; (2) substantial conditioning appears necessary; and (3) early diagnosis and absence of opportunistic infections may affect outcome.

SummaryCommon variable immunodeficiency disorders (CVIDs) are a heterogeneous group of diseases characterized by hypogammaglobulinaemia and consequent susceptibility to infection. CVID patients commonly develop a variety of additional manifestations for which the causative factors are not fully understood. Two such manifestations are granulomatous disease and enteropathy. Because the ability to predict complications would aid clinical management, we continue to search for possible disease modifier genes. NOD2 acts a microbial sensor and is involved in proinflammatory signalling. Particular mutations of the NOD2 gene are associated with Crohn's disease including gly908arg, leu1007finsc and arg702trp polymorphisms. We hypothesized that NOD2 polymorphisms may be a disease modifier gene towards an enteropathic or granulomatous phenotype within CVIDs. Sequence-specific primers returned genotypes for 285 CVID patients from centres across the United Kingdom and Europe. We present the frequencies of the different phenotypes of patients within our international cohort. Arg702trp polymorphisms were significantly less frequent than wild-type (WT) (P = 0·038) among international CVID patients with splenomegaly. Gly908arg polymorphisms were more prevalent than WT in UK patients with autoimmune disorders (P = 0·049) or enteropathy (P = 0·049). NOD2 polymorphisms were not more prevalent than WT in CVID patients with clinical phenotypes of granulomata. UK allele frequencies of 0·014, 0·056 and 0·026 were found for gly908arg, arg702trp and leu1007finsc NOD2 polymorphisms, respectively. These do not differ significantly from UK immunocompetent controls confirming, as expected, that in addition these NOD2 polymorphisms do not confer susceptibility to CVIDs per se.

Summary X-linked agammaglobulinaemia (XLA) is characterized by absence of mature B cells because of mutations in the Bruton's tyrosine kinase (Btk) gene. Btk-deficient early B cell precursors experience a block in their differentiation potentially reversible by the addition of an intact Btk gene. Btk expression was measured in 69 XLA patients with 47 different mutations and normal expression was detected in seven. We characterized these Btk mutant forms functionally by transfection into a lymphoma cell line that lacks endogenous Btk expression (Btk−/− DT40 cells) and analysed the calcium flux in response to B cell receptor stimulation. To test whether co-expression of a mutated form could compromise the function of the intact Btk transfection, studies in wild-type (WT) DT40 cells were also performed. Study reveals that none of the seven Btk mutants analysed was able to revert the absence of calcium mobilization upon IgM engagement in Btk−/− DT40 cells, as does intact Btk. In addition, calcium mobilization by anti-IgM stimulation in DT40 Btk+/+ cells was unaffected by co-expression with Btk mutants. These results suggest that gene addition would be feasible not only for patients with XLA and mutations that prevent Btk expression, but for those with expression of a mutant Btk.

Background X-linked agammaglobulinemia (XLA) is characterized by impaired B-cell differentiation caused by mutations in the Bruton's tyrosine kinase (Btk) gene. The natural disease model, the X-linked immunodeficiency mouse, shows a less severe phenotype, indicating a different requirement of Btk in human and mouse B cells. Btk is also expressed in the myeloid line and participates in LPS signaling. Deficient oxidative burst and myeloid differentiation have been reported in the X-linked immunodeficiency mouse, but the precise mechanism and relevance of Btk activity in human monocytes is poorly understood. Objective The apparent absence in XLA of clinical manifestations of myeloid deficiency prompted us to explore the relevance of complete Btk absence in human myeloid cells. Methods Seven patients with XLA with BTK mutations conditioning a null protein expression were included in the study. Monocyte LPS-induced mitogen-activated protein kinase activation, TNF-α and IL-6 production in monocytes, and oxidative burst in monocytes and granulocytes were analyzed by means of flow cytometry. Results We show that in response to LPS, Btk-null monocytes from patients with XLA induce early mitogen-activated protein kinase activation and intracellular TNF-α and IL-6 production with the same intensity as cells from age- and sex-matched control subjects. In addition, the oxidative burst in response to LPS and other stimulants was completely normal in Btk-null monocytes and neutrophils. Conclusion Our results indicate that Btk is not essential for early LPS signaling in human monocytes and that different Btk dependency might exist between human and mouse myeloid cells. Clinical implications These findings provide a better understanding of XLA, and they show the differences between human XLA and murine Xid models.

Background Common variable immunodeficiency disorders (CVIDs) are the most common forms of symptomatic primary antibody failure in adults and children. Replacement immunoglobulin is the standard treatment, although there are few consistent data on optimal dosages and target trough IgG levels required for infection prevention. Objective To provide data to support the hypothesis that each patient requires an individual dose of therapeutic immunoglobulin to prevent breakthrough infections and that efficacious trough IgG levels vary between patients. Methods Data, collected prospectively from a cohort of 90 patients with confirmed CVIDs from 1 center over a follow-up period of 22 years, was validated and analyzed. Immunoglobulin doses had been adjusted in accordance with infections rather than to achieve a particular trough IgG level. Doses to achieve infection-free periods were determined and resultant trough levels analyzed. A smaller group of patients with X-linked agammaglobulinemia was analyzed for comparison. Results Patients with a CVID had a range of trough IgG levels that prevented breakthrough bacterial infections (5-17 g/L); viral and fungal infections were rare. Doses of replacement immunoglobulin to prevent breakthrough infections ranged from 0.2 to 1.2 g/kg/mo. Those with proven bronchiectasis or particular clinical phenotypes required higher replacement doses. Patients with X-linked agammaglobulinemia showed a similar range of IgG levels to stay infection-free (8-13 g/L). Conclusion These data offer guidance regarding optimal doses and target trough IgG levels in individual patients with CVIDs with or without bronchiectasis and for particular clinical phenotypes. The goal of replacement therapy should be to improve clinical outcome and not to reach a particular IgG trough level.

Background X-linked (Bruton's) agammaglobulinemia (XLA) is a rare immunodeficiency caused by a block in B-cell development caused by mutations in the Bruton's tyrosine kinase (BTK) gene. Many aspects of XLA and BTK function remain unresolved; atypical presentations have been reported, and no clear genotype-phenotype correlation has been established. Objectives We sought to contribute to the understanding of XLA through the phenotypic and biochemical characterization of a large group of Spanish patients with agammaglobulinemia. We also sought to classify the mutations according to their severity to analyze a genotype-phenotype correlation. Methods Clinical and analytic data were collected from the clinical records. We studied the BTK gene, protein expression, and function, and the findings were correlated with the phenotypic information. Results Fifty-four patients were given diagnoses of XLA. We identified 38 different mutations in BTK, 26 not described in other patients, and several uncommon clinical phenotypes or analytic characteristics were found. The statistical analysis shows that less severe mutations or minimal detection of protein by means of flow cytometry are associated with decreased severity in clinical and analytic data, demonstrating a clear relation between the type of mutation and the disease expressivity. However, some exemptions to this rule were noted. Conclusions XLA is a variable disease. Globally, a genotype-phenotype correlation is observed, but individual discrepancies between the severity of the mutation and the clinical and analytic phenotype suggest that other loci or ambient factors significantly influence the disease presentation and evolution.